EP3476933A1 - Dreidimensional gezüchteter hautlappen, zur herstellung davon verwendeter zellzüchtungsbehälter und verfahren zur herstellung eines dreidimensional gezüchteten hautlappens - Google Patents

Dreidimensional gezüchteter hautlappen, zur herstellung davon verwendeter zellzüchtungsbehälter und verfahren zur herstellung eines dreidimensional gezüchteten hautlappens Download PDF

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EP3476933A1
EP3476933A1 EP17815530.5A EP17815530A EP3476933A1 EP 3476933 A1 EP3476933 A1 EP 3476933A1 EP 17815530 A EP17815530 A EP 17815530A EP 3476933 A1 EP3476933 A1 EP 3476933A1
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Prior art keywords
skin sheet
convex sections
dimensional culture
cells
average
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English (en)
French (fr)
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EP3476933A4 (de
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Mitsuhiro Denda
Sumiko Denda
Junichi KUMAMOTO
Yasuaki Kobayashi
Masaharu NAGAYAMA
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Shiseido Co Ltd
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Shiseido Co Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L1/00—Enclosures; Chambers
    • B01L1/02—Air-pressure chambers; Air-locks therefor
    • B01L1/025—Environmental chambers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
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    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2513/00—3D culture
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
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    • C12N2535/00—Supports or coatings for cell culture characterised by topography

Definitions

  • the present invention relates to a three-dimensional culture skin sheet.
  • the invention further relates to a cell culturing vessel for use in production of a three-dimensional culture skin sheet.
  • the invention still further relates to a method for preparing a three-dimensional culture skin sheet.
  • the present application has been filed claiming the priority of a Japanese application filed on June 24, 2016 (Japanese Patent Application No. 2016-125843 ) and a Japanese application filed on December 28, 2016 (Japanese Patent Application No. 2016-256778 ).
  • the skin is an organ that covers the surface of the body, separating the interior of the body from its exterior. Skin functions as a physical barrier and protects the interior of the body from dryness and infiltration of hazardous substances, thus performing an indispensable role for life maintenance.
  • the skin of higher vertebrates primarily consists of the epidermis, dermis and subcutaneous tissue layers, in order from the outermost layer.
  • the interface between the epidermis and the dermis is corrugated, and the corrugations are known to flatten with age (see NPL 1, for example).
  • the epidermis is composed mainly of cells known as keratinocytes, the keratinocytes dividing at the deepest part (basal lamina) of the epidermis while migrating to the surface and differentiating toward the upper layer into the stratum spinosum, granular layer and stratum corneum, eventually being shed as old skin. This cycle takes about 4 weeks in humans.
  • Such cultured skin models are used for drug safety testing or basic research for cosmetic products or skin-applied drugs, and they are garnering increasing interest as animal-substituting models, but a cultured skin model having the same level of thickness and barrier function as human skin has not yet been obtained.
  • the present invention encompasses the following inventions.
  • the three-dimensional culture skin sheet of the invention it is possible to obtain a base section (epidermal basal membrane-like structure) having concavoconvexities that have not been present in three-dimensional culture skin sheets of the prior art, as well as a thickened three-dimensional culture skin sheet. It is thereby possible to provide a three-dimensional skin model that is useful for safety testing and basic research for cosmetics or skin-applied drugs. Furthermore, using the cell culturing vessel of the invention, it is possible to provide, in a stable and economical manner, a thickened three-dimensional culture skin sheet which has not been obtainable in the prior art. Using the culturing method of the invention, as well, it is possible to provide, in a stable and economical manner, a thickened three-dimensional culture skin sheet which has not been obtainable in the prior art.
  • Fig. 1 is a cross-sectional view showing a cell culturing vessel 1 to be used for this embodiment, and a cross-sectional magnified view 6 of a portion of the culture surface of the same.
  • the cell culturing vessel 1 includes a cell culture insert 2 in which the cells are seeded, and a bottom well 4 for filling with culture medium around the outside of the cell culture insert 2.
  • the cell culture insert 2 comprises a porous film 3.
  • the nutrients and oxygen can be supplied from the culture medium 5 filling the outside of the cell culture insert 2 through the porous film 3 to the cells on the porous film 3.
  • the surface of the porous film 3 comprises convex sections 7 for formation of concavoconvexities on the three-dimensional culture skin sheet 10 of the invention.
  • the epidermal cells become overlaid, and it is possible to obtain a three-dimensional culture skin sheet 10 having concavoconvexities in at least one portion of the base section 11 of the three-dimensional culture skin sheet 10 (the bold lines in Fig. 2 ), and/or an epidermal cell-free region in at least one portion of the interior of the three-dimensional culture skin sheet.
  • the "base section" of the three-dimensional culture skin sheet 10 is the side of the epidermal cells formed as a sheet that contacts with the surface of the cell culturing vessel 1 when the epidermal cells have been seeded in the cell culturing vessel, and for example, it is the side in contact with the surface of the porous film 3 shown in Fig. 1 and/or with the convex sections 7 (11 and the bold lines in Fig. 2 ).
  • the "base section” is a structure corresponding to the basal membrane formed between the epidermis and dermis in living skin tissue, it does not necessarily need to have the same membrane structure as a living basal membrane.
  • the “base section” for the invention includes a "epidermal basal membrane-like structure” having a concavoconvex shape in at least one portion, the epidermal basal membrane-like structure including epidermal cells.
  • the epidermal basal membrane-like structure may also include a basal membrane as formed by epidermal cells in living skin tissue.
  • Fig. 2 is a cross-sectional view of a three-dimensional culture skin sheet 10 having the porous film 3 and convex sections 7 of Fig. 1(b) removed.
  • the three-dimensional culture skin sheet 10 includes a stratum corneum 9 which lacks cell nuclei, and epidermal cells 8 in addition to the stratum corneum 9. Due to the convex sections 7 present on the porous film 3, the three-dimensional culture skin sheet 10 has concavoconvexities formed in a manner with cells surrounding epidermal cell-free regions Va and/or epidermal cell-free regions Vb in the epidermal cell-free region inside the three-dimensional culture skin sheet.
  • concavoconvexities in the base section 11 of the three-dimensional culture skin sheet 10 are formed in at least one portion of the side of the three-dimensional culture skin sheet of the invention that is in contact with the culture surface of the cell culturing vessel, and the term refers to undulations that are larger than undulations accidentally obtained when epidermal cells have been cultured on the surface of a flat culturing instrument.
  • the convexities of the concavoconvexities of the three-dimensional culture skin sheet 10 are portions of the base section 11 that protrude toward the direction of air exposure (the upper layer) of the cell culturing vessel, and for example, they are the convex sections T1 of the three-dimensional culture skin sheet in Fig.
  • the concavities of the concavoconvexities of the three-dimensional culture skin sheet are the sections that are depressed in the direction of the base section of the epidermis, opposite from the convexities, and for example, they are the recesses B1 of the three-dimensional cell sheet in Fig. 2 .
  • the distance between the tip of any convex section T1 of the three-dimensional culture skin sheet and the maximum depression of a recess B1 of the three-dimensional culture skin sheet adjacent to that tip, in the direction perpendicular to the culture surface, may be considered to be the height H'1 of the concavoconvexities of the three-dimensional culture skin sheet.
  • the form and height H'1 of the concavoconvexities formed in the three-dimensional culture skin sheet 10 depend on the form and height H of the convex sections 7 on the porous film 3.
  • the separation width W'1 between convex sections of the three-dimensional culture skin sheet formed in the three-dimensional culture skin sheet 10 depends on the separation width W between the convex sections formed by the convex sections 7 on the porous film 3.
  • the width Y' of the convex sections of the three-dimensional culture skin sheet formed in the three-dimensional culture skin sheet 10 depends on the width Y of the convex sections formed by the convex sections 7 on the porous film 3.
  • the height H'1 of the concavoconvexities of the three-dimensional culture skin sheet, the separation width W'1 between the convex sections of the three-dimensional culture skin sheet and/or the width Y' of the convex sections of the three-dimensional culture skin sheet may be measured, for example, using a commercially available optical microscope with preparation of an HE-stained tissue section, or they may be measured by observation using a fluorescent microscope, confocal microscope or two-photon laser microscope, with immunohistochemical staining, or they may be measured by observation using an electron microscope, without any particular restrictions.
  • the height H'1 of the concavoconvexities of the three-dimensional culture skin sheet can be measured from an image taken using a camera-equipped apparatus such as a common microscope or fluorescent microscope.
  • the microscope used may be, for example, a BX51 system industrial (transmission/reflection) microscope by Olympus Corp.
  • the camera used may be, for example, a DP71 microscope digital camera by Olympus Corp.
  • the taken image may be loaded into computer analysis software and analyzed by an established image analysis method.
  • a portion of any cross-section of the three-dimensional culture skin sheet 10 will sometimes have an epidermal cell-free region Vb where no concavoconvexities are formed in the base section 11 of the three-dimensional culture skin sheet 10, as shown at right in Fig. 2 . Since cells are present in the lower region of the bottom section B2 of the epidermal cell-free region of the epidermal cell-free region Vb of the cross-section, when viewing the cross-section it may appear not to form a concavoconvex shape connected with the outside of the three-dimensional culture skin sheet 10.
  • an epidermal cell-free region Vb may be observed in a cross-section where the convex sections 7 are not in contact with the porous film 3.
  • an epidermal cell-free region Vb may be observed in a cross-section including a location where fibers are not bonded to the porous film 3.
  • An epidermal cell-free region Vb may also be observed when the convex sections 7 are free from the top of the porous film 3 in the cell culture and cells infiltrate and engraft onto the bottom parts of the convex sections 7.
  • the concavoconvexities of the three-dimensional culture skin sheet 10 include shapes where the epidermal cells 8 surround epidermal cell-free regions Vb that include the examples mentioned above. In most cases the height H'2 of the epidermal cell-free region Vb approximately matches the height H'1 of the concavoconvexities of the three-dimensional culture skin sheet.
  • the term three-dimensional means that the cells are stacked in the vertical direction to form an overlaid state, which differs from an approximately single cell layer obtained by culturing conventional adherent cells in a cell culture dish or the like, i.e. a two-dimensional cell layer form.
  • the three-dimensional culture skin sheet of the invention has concavoconvexities in an epidermal basal membrane-like structure, as a three-dimensionalized structure with thickness, in which the epidermal cells are overlaid.
  • the term "three-dimensional culture skin sheet” is distinguished from skin tissue inherently found in living bodies, and refers to three-dimensional skin-like tissue obtained by seeding a cell group including cells from skin tissue obtained by degrading intercellular adhesion proteins to separate them, and/or skin-forming cells obtained by inducing differentiation from pluripotent stem cells, in a cell culturing vessel and culturing them on the cell culturing vessel, to reconstitute the cells into a sheet.
  • the cells forming the three-dimensional culture skin sheet include epidermal cells, and they may be considered to be a three-dimensional culture epidermal sheet.
  • the three-dimensional culture skin sheet or three-dimensional culture epidermal sheet may also include cells other than epidermal cells, such as cells other than epidermal cells that are components of the epidermis (such as melanocytes, Langerhans cells and Merkel cells), and/or cells present in dermal tissue (fibroblasts, neurons, mast cells, plasmocytes, vascular endothelial cells, histiocytes and Meissner corpuscles).
  • the three-dimensional culture skin sheet of the invention may also be one comprising a porous film 3 having convex sections in contact with the base section 11.
  • the cells composing the three-dimensional culture skin sheet of the invention may be derived from any animal, but they are preferably derived from a vertebrate, more preferably from a mammal, and most preferably from a human.
  • epidermal cells means cells including all cells composing the epidermis at different stages of differentiation.
  • Epidermal cells mainly consist of cells known as keratinocytes.
  • epidermal tissue is formed of epidermal cells at different stages of differentiation that are overlaid in a laminar fashion.
  • the deepest portion of the epidermis is called the basal lamina or basal cell layer (hereunder referred to as “basal lamina”), where cylindrical cells form a single layer and are thought to include stem cells.
  • the basal lamina is also an interface with the dermis, and the stratum spinosum is present on its upper layer.
  • a papillary layer forming the dermis is present directly under the basal lamina.
  • the stratum spinosum is also known as the squamous cell layer, and is composed of approximately 2 to 10 layers in biological tissue.
  • the layer is called the "stratum spinosum” because interconnected "spines" are observed.
  • Only cells of the basal lamina have proliferation potency, and with progressive stages of differentiation, epidermal cells migrate to the outer layer while changing to a flat form.
  • the differentiation stage progresses from the stratum spinosum, a granular layer with keratohyalin granules and lamellar granules (granulocyte layer) is formed.
  • the granular layer is composed of about 2 or 3 layers in biological tissue.
  • stratum corneum stratum corneum cell layer
  • the epidermis is largely composed of these 4 types of layers, but epidermal tissue also includes melanocytes, Langerhans cells and Merkel cells in addition to epidermal cells, each of which prevent ultraviolet rays from reaching the dermis, perform an important role in skin immunological function, and contribute to sensation.
  • the three-dimensional culture skin sheet may contain cells other than epidermal cells, and it may be modified as appropriate according to the purpose of use.
  • the "barrier function" of skin generally refers to the function of preventing loss of moisture or biological components from the body, or the function of preventing entrance of foreign matter (microorganisms, viruses and dust) into the body from the exterior.
  • the barrier function of the three-dimensional culture skin sheet of the invention can be evaluated by examining the transepidermal water loss (TEWL). Lower transepidermal water loss, or moisture permeation, from the three-dimensional culture skin sheet represents a higher barrier function.
  • the method used to examine the transepidermal water loss may be a method commonly employed by those skilled in the art (see Kumamoto J., Tsutsumi M., Goto M., Nagayama M., Denda M., Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo. Arch. Dermatol. Res. 308:49-54, 2016 , for example).
  • the average lower limit for the height H'1 of the concavoconvexities of the three-dimensional culture skin sheet may be 1 ⁇ m or greater, 5 ⁇ m or greater, 10 ⁇ m or greater, 15 ⁇ m or greater, 20 ⁇ m or greater, 25 ⁇ m or greater or 30 ⁇ m or greater, for example.
  • the average upper limit for the height H'1 of the concavoconvexities of the three-dimensional culture skin sheet may be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 150 ⁇ m or smaller, 100 ⁇ m or smaller, 80 ⁇ m or smaller or 75 ⁇ m or smaller.
  • the average for the height H'1 of the concavoconvexities of the three-dimensional culture skin sheet may be 1 ⁇ m to 300 ⁇ m, 1 ⁇ m to 200 ⁇ m, 1 ⁇ m to 100 ⁇ m, 5 ⁇ m to 250 ⁇ m, 10 ⁇ m to 200 ⁇ m, 15 ⁇ m to 150 ⁇ m, 20 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the average lower limit for the separation width W'1 (or W'2) between the convex sections of the three-dimensional culture skin sheet of the may be 1 ⁇ m or greater, 5 ⁇ m or greater, 8 ⁇ m or greater, 10 ⁇ m or greater, 12 ⁇ m or greater, or 15 ⁇ m or greater, for example.
  • the average upper limit for the separation width W'1 (or W'2) between the convex sections of the three-dimensional culture skin sheet may be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 180 ⁇ m or smaller, 170 ⁇ m or smaller, 150 ⁇ m or smaller, 125 ⁇ m or smaller, 100 ⁇ m or smaller or 90 ⁇ or smaller, for example.
  • the average for the separation width W'1 (or W'2) between the convex sections of the three-dimensional culture skin sheet may be 1 ⁇ m to 300 ⁇ m, 1 ⁇ m to 200 ⁇ m, 1 ⁇ m to 100 ⁇ m, 1 ⁇ m to 90 ⁇ m, 5 ⁇ m to 250 ⁇ m, 8 ⁇ m to 200 ⁇ m, 10 ⁇ m to 180 ⁇ m, 12 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the average lower limit for the width Y'1 of the convex sections of the three-dimensional culture skin sheet may be 1 ⁇ m or greater, 5 ⁇ m or greater, 10 ⁇ m or greater, 15 ⁇ m or greater, 20 ⁇ m or greater or 25 ⁇ m or greater, for example.
  • the average upper limit for the width Y'1 of the convex sections of the three-dimensional culture skin sheet may be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 150 ⁇ m or smaller, 100 ⁇ m or smaller, 80 ⁇ m or smaller or 75 ⁇ m or smaller, for example.
  • the average for the width Y'1 of the convex sections of the three-dimensional culture skin sheet may be 1 ⁇ m to 300 ⁇ m, 5 ⁇ m to 250 ⁇ m, 10 ⁇ m to 200 ⁇ m, 15 ⁇ m to 150 ⁇ m, 20 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • a three-dimensional culture skin sheet with thicker epidermal cell layers on the base section 11 will be obtained, compared to one without concavoconvexities.
  • the three-dimensional culture skin sheet obtained by the invention has an excellent effect of thickening simply by physical formation of concavoconvexities in the base section 11 by convex sections formed of non-biological substances, instead of relying on convex sections composed of cells and biological substances such as cell adhesion proteins.
  • the convex sections are convex sections composed of non-biological substances, it is possible to stably maintain the concavoconvex structure on the base section of the three-dimensional culture skin sheet without degradation of the convex sections, whether used during the culturing period or afterwards, and to thus continuously maintain the effect of the invention.
  • the average height, shapes and separation width of the concavoconvexities of the three-dimensional culture skin sheet of the invention can be controlled by the heights, shapes and separation width of the convex sections formed in the porous film surface of the cell culturing vessel.
  • the three-dimensional culture skin sheet according to the invention is a three-dimensional culture skin sheet with low transepidermal water loss and a high barrier function compared to a conventional three-dimensional culture skin sheet.
  • the barrier function is improved by the presence of the concavoconvexities in at least one portion of the base section 11 of the three-dimensional culture skin sheet.
  • the base section 11 of the three-dimensional culture skin sheet is preferably closely bonded with the culture surface (the culture surface including the porous film and the convex sections).
  • the barrier function is further improved if the base section 11 of the three-dimensional culture skin sheet is closely bonded with the culture surface.
  • the base section 11 of the three-dimensional culture skin sheet is more preferably in a closely bonded state with the entire culture surface.
  • the convex sections provided in the surface of the porous film may be composed of biological substances, or composed of non-biological substances, or composed of a combination of biological substances and non-biological substances.
  • non-biological substances means substances excluding biological substances, i.e. biopolymers that constitute living bodies (nucleic acids, proteins, polysaccharides and their constituent elements (nucleotides, nucleosides, amino acids and sugars), and vitamins.
  • the non-biological substance used to form the convex sections of the invention is preferably a biocompatible substance that does not affect culturing of the cells.
  • the convex sections provided on the surface of the porous film may be further coated on their surfaces with a biological substance such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (the D-form or L-form) or thrombospondin.
  • a biological substance such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (the D-form or L-form) or thrombospondin.
  • the cells to be used for the invention may be primary epidermal cells obtained by finely chopping biological tissue, and especially skin tissue, and treating it with a protease such as collagenase or trypsin, or they may be epidermal cells obtained by subculturing primary epidermal cells to cause their proliferation, or epidermal cells obtained by inducing differentiation from pluripotent stem cells such as ES cells, iPS cells or Muse cells, or they may be epidermal cells from an established line.
  • they are primary cultured epidermal cells obtained by harvesting from biological tissue and seeding, or subcultured epidermal cells obtained by further subculturing the primary cultured cells. If they are epidermal cells obtained by harvesting from biological tissue, they will be more likely to maintain the same properties as in the body, which is convenient when conducting tests for examining drug efficacy and side-effects, or for use in basic research.
  • the primary cultured epidermal cells are epidermal cells that have been harvested from biological tissue and then cultured only once in a cell culturing vessel and collected, and they may be referred to as "passage number: 0 (or first-generation)" epidermal cells.
  • the non-subcultured epidermal cells that have become confluent or subconfluent may be subcultured by a method known to those skilled in the art, and further subjected to amplification culturing.
  • Epidermal cells obtained by a single subculturing procedure are referred to as "passage number: 1" (or second-generation)" epidermal cells.
  • n (where n (integer) is the passage number) (n + 1 generations) may also be used, corresponding to the number of subcultures.
  • Commercially available primary cultured cells for example, frozen NHEK (NB), Catalog No.: KK-4009 by Kurabo Industries, Ltd.
  • NB frozen NHEK
  • a step of freezing the cells may also be included between each subculturing procedure.
  • somatic cell lines Since most established somatic cell lines usually undergo changes in properties or variation in the proportion of constituent cells with repeated subculturing, they are altered into a cell population that is different from the cell population with a low number of passages. This is thought to result because the cells experience senescence due to shortening of telomeres with each cell division during repeated subculturing, and due to biological stress and/or physical stress resulting from confluency and/or trypsin treatment and the like. Usually, therefore, when using a tissue model comprising cultured cells, the cells are used with a lower number of passages.
  • primary cultured cells are cells directly isolated from biological tissue and have a low number of passages, there are limits to the number of cells that can be supplied.
  • the sizes and amounts of tissue that can be harvested are not only limited by ethical considerations, but variations also exist in the quality of the cells that can be obtained by donors.
  • primary cultured cells are commercially available, they are necessarily expensive due to supply volume issues. When it is necessary to construct a large three-dimensional culture skin sheet, therefore, it is necessary to obtain and use expensive cells, which raises a problem in terms of cost.
  • primary cultured cells that have been subcultured and proliferated may be used, but as mentioned above, the obtained cells are not necessarily limited to those exhibiting the same properties as primary cultured cells.
  • Three-dimensional cultured skin model of epidermal cells for example, keratinocyte three-dimensional culture starter kit by CELLnTEC
  • the three-dimensionalizing nature spontaneously weakens, making it impossible to obtain a thick three-dimensional cultured skin model (also known as a three-dimensional culture skin sheet) (the controls of Examples 1 to 3 described below are examples of this).
  • a three-dimensional culture skin sheet having a thickness that has only been obtainable in the prior art with primary cultured cells is constructed even when using epidermal cells with a passage number of 2 or greater after isolated culture from biological tissue. Even when primary cultured cells (for example, passage number: 0 or 1) are used, a thickened three-dimensional culture skin sheet according to the invention can be obtained.
  • the passage number of the epidermal cells used after isolated culture from biological tissue may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, for example, with no limitation so long as the stratum spinosum, granular layer and/or stratum corneum of the three-dimensional culture skin sheet are thickened.
  • the animal species of the primary epidermal cells used is not particularly restricted but is preferably a human.
  • the primary epidermal cells used may be from a fetus, neonate, juvenile or adult, but they are preferably cells from a fetus, neonate or juvenile. When cells from a human juvenile are used, the cells may be of age younger than 20, such as 1 to 19, 1 to 10 or 1 to 5 years of age. Even when cells from a human adult are used, the cells used are preferably younger, such as 20 to 29, 30 to 39 or 40 to 49 years of age.
  • the average thickness of the three-dimensional culture skin sheet may be 20 ⁇ m or greater, 21 ⁇ m or greater, 22 ⁇ m or greater, 23 ⁇ m or greater, 24 ⁇ m or greater, 25 ⁇ m or greater, 26 ⁇ m or greater, 27 ⁇ m or greater, 28 ⁇ m or greater, 29 ⁇ m or greater, 30 ⁇ m or greater, 35 ⁇ m or greater, 40 ⁇ m or greater, 45 ⁇ m or greater, 50 ⁇ m or greater, 55 ⁇ m or greater, 60 ⁇ m or greater, 65 ⁇ m or greater, 70 ⁇ m or greater, 75 ⁇ m or greater, 80 ⁇ m or greater, 85 ⁇ m or greater, 90 ⁇ m or greater, 95 ⁇ m or greater, 100 ⁇ m or greater, 105 ⁇ m or greater, 110 ⁇ m or greater, 115 ⁇ m or greater, 120 ⁇ m or greater, 125 ⁇ m or greater, 150 ⁇ m or greater, 175 ⁇ m or greater, 200 ⁇ m or greater,
  • Fig. 1 shows a conceptual drawing of a cell culturing vessel for creation of a three-dimensional culture skin sheet of the invention. Modified examples of the convex sections 7 in the cell culturing vessel 1 are shown in Fig. 3 to Fig. 5 .
  • Fig. 3 shows an embodiment of a cell culturing vessel 1 of the invention.
  • convex sections 71 are arranged for formation of concavoconvexities in the three-dimensional culture skin sheet of the invention.
  • the convex sections 71 have bead shapes.
  • the convex sections 71a may be arranged at the locations of square lattice points.
  • the separation width W1a between the convex sections it is possible to control the separation width W'1 between the convex sections in the three-dimensional culture skin sheet of the invention.
  • the convex sections 71b may be arranged at the locations of regular triangular lattice points.
  • the separation width W1b between the convex sections it is possible to control the separation width W'1 between the convex sections in the three-dimensional culture skin sheet of the invention.
  • the width Y1 of the convex sections it is possible to control the width Y' of the recesses in the three-dimensional culture skin sheet of the invention.
  • Fig. 4 shows an embodiment of a cell culturing vessel 1 of the invention.
  • convex sections 72 are arranged for formation of concavoconvexities in the three-dimensional culture skin sheet of the invention.
  • the convex sections 72 have cubic shapes, as types of rectangular columnar shapes.
  • the convex sections 72a may be arranged at the locations of square lattice points.
  • the separation width W2a between the convex sections By adjusting the separation width W2a between the convex sections, it is possible to control the separation width W'1 between the convex sections in the three-dimensional culture skin sheet of the invention.
  • the centers of gravity of the convex sections 72b may be arranged at the locations of regular triangular lattice points.
  • the separation width W2b between the convex sections By adjusting the separation width W2b between the convex sections, it is possible to control the separation width W'1 between the convex sections in the three-dimensional culture skin sheet of the invention.
  • the width Y2 of the convex sections it is possible to control the width Y' of the recesses in the three-dimensional culture skin sheet of the invention.
  • Fig. 5 is a cross-sectional view showing an embodiment of convex sections (73, 74, 75) provided on a cell culturing vessel 1 of the invention.
  • Fig. 5(a) shows convex sections 73 with pyramidal shapes (triangular pyramids, square pyramids or circular cones).
  • Fig. 5(b) shows convex sections 74 with frustum shapes.
  • Fig. 5(c) shows convex sections 75 with bell shapes.
  • the shapes of the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are not limited to those described above, and include approximately semi-spherical shapes and approximately cuboid shapes, for example.
  • the material of the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be composed of biological substances, or composed of non-biological substances, or composed of a combination of biological substances and non-biological substances.
  • PET polyethylene terephthalate
  • the surfaces of the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may also be subjected to plasma treatment by a publicly known method, in order to facilitate adhesion of the cells.
  • the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be arranged at equal spacings on the porous film 3 as in the embodiments described above, or they may be arranged at non-uniform spacings, with an average lower limit for the spacings being 1 ⁇ m or greater, 5 ⁇ m or greater, 8 ⁇ m or greater, 10 ⁇ m or greater, 12 ⁇ m or greater or 15 ⁇ m or greater, for example.
  • the average upper limit for the spacings between the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may also be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 180 ⁇ m or smaller, 170 ⁇ m or smaller, 150 ⁇ m or smaller, 125 ⁇ m or smaller or 100 ⁇ m or smaller, for example.
  • the average spacing between the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be 1 ⁇ m to 300 ⁇ m, 5 ⁇ m to 250 ⁇ m, 8 ⁇ m to 200 ⁇ m, 10 ⁇ m to 180 ⁇ m, 12 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the average lower limit for the heights (H, HI, H2, H3, H4, H5) of the convex sections may be 1 ⁇ m or greater, 5 ⁇ m or greater, 10 ⁇ m or greater, 15 ⁇ m or greater, 20 ⁇ m or greater, 25 ⁇ m or greater or 30 ⁇ m or greater, for example.
  • the average upper limit for the heights (H, H2, H3, H4, H5) of the convex sections may also be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 150 ⁇ m or smaller, 100 ⁇ m or smaller, 80 ⁇ m or smaller or 75 ⁇ m or smaller, for example.
  • the average of the heights (H, H2, H3, H4, H5) of the convex sections may be 1 ⁇ m to 300 ⁇ m, 5 ⁇ m to 250 ⁇ m, 10 ⁇ m to 200 ⁇ m, 15 ⁇ m to 150 ⁇ m, 20 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the average lower limit for the widths (Y, Y1, Y2, Y3, Y4, Y5) between the convex sections may be 1 ⁇ m or greater, 5 ⁇ m or greater, 10 ⁇ m or greater, 15 ⁇ m or greater, 20 ⁇ m or greater or 25 ⁇ m or greater, for example.
  • the average upper limit for the widths of the convex sections (Y, Y1, Y2, Y3, Y4, Y5) may be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 150 ⁇ m or smaller, 100 ⁇ m or smaller, 80 ⁇ m or smaller or 75 ⁇ m or smaller, for example.
  • the average for the widths (Y, Y1, Y2, Y3, Y4, Y5) of the convex sections may be 1 ⁇ m to 300 ⁇ m, 5 ⁇ m to 250 ⁇ m, 10 ⁇ m to 200 ⁇ m, 15 ⁇ m to 150 ⁇ m, 20 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be anchored onto the porous film 3 in aggregates of several or several dozen or more.
  • the method of anchoring the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be anchoring onto the porous film 3 in a covalently bonded manner by a publicly known method, or anchoring using an adhesive.
  • the adhesive is preferably a substance that does not affect cell adhesion and has no toxicity for cells, and that is not degraded by the cells.
  • Examples include poly-D-lysine and agarose, but any other adhesives may be used so long as they are substances that produce a similar effect and are able to anchor the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) onto the porous film 3.
  • the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may even be formed directly on the porous film 3, and for example, they may be formed by a method using 3D printer technology or semiconductor process technology (for example, a method combining a lithography process (including nanoimprinting or photoembossing) and dry etching (for example, reactive ion etching) or wet etching).
  • the convex sections (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be simply placed on the porous film 3.
  • Fig. 1 shows a conceptual drawing of a cell culturing vessel for creation of a three-dimensional culture skin sheet of the invention. Additional modified examples of the convex sections 7 in the cell culturing vessel 1 are shown in Figs. 6 and 7 .
  • Fig. 6 shows an embodiment of a cell culturing vessel 1 of the invention.
  • convex sections (76, 77, 78) are provided for formation of concavoconvexities in the three-dimensional culture skin sheet of the invention.
  • the convex sections 76 may be arranged in an approximately parallel striped manner on the porous film 3.
  • the convex sections 77 may be arranged in a lattice-like manner with equal spacings in the longitudinal and transverse directions on the porous film 3.
  • Fig. 6 shows an embodiment of a cell culturing vessel 1 of the invention.
  • convex sections 76, 77, 78
  • the convex sections 77 may be arranged in a lattice-like manner with equal spacings in the longitudinal and transverse directions on the porous film 3.
  • the convex sections 78 may be arranged as a regular triangular lattice on the porous film 3.
  • the convex sections (76, 77, 78) may be formed by fibers, for example.
  • the convex sections (76, 77, 78) may be formed of single fibers, or they may be formed of bundles of multiple narrow fibers.
  • the convex sections (76, 77, 78) may be formed as cuboid structures, or they may be constructed of approximately semicircular structures.
  • Fig. 7 shows an embodiment according to Fig. 6(b) , which is a cell culturing vessel 1 comprising convex sections composed of alternately knitted convex sections 77a and convex sections 77b.
  • Fig. 7(b) shows a cross-section on the line connecting Z and Z' in Fig. 7(a) .
  • the material of the convex sections may be composed of biological substances, or composed of non-biological substances, or composed of a combination of biological substances and non-biological substances.
  • examples include polyesters or polyethylene terephthalate (PET), polycarbonate, polystyrene, glass, insoluble collagen, silicon rubber and the like, or other materials with no particular restrictions so long as the cells can adhere and culturing is possible.
  • the average lower limit for the heights of the convex sections may be 1 ⁇ m or greater, 5 ⁇ m or greater, 10 ⁇ m or greater, 15 ⁇ m or greater, 20 ⁇ m or greater, 25 ⁇ m or greater or 30 ⁇ m or greater, for example.
  • the average upper limit for the heights of the convex sections may also be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 150 ⁇ m or smaller, 100 ⁇ m or smaller, 80 ⁇ m or smaller or 75 ⁇ m or smaller, for example.
  • the average of the heights of the convex sections may be 1 ⁇ m to 300 ⁇ m, 5 ⁇ m to 250 ⁇ m, 10 ⁇ m to 200 ⁇ m, 15 ⁇ m to 150 ⁇ m, 20 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the height H7a of the convex sections (77a, 77b) can be determined based on the uppermost sections (C, C') of the raised intersections where the convex sections 77a and convex sections 77b cross.
  • the convex sections (76, 77, 77a, 77b, 78) may be anchored at equal spacings or anchored at non-uniform spacings on the porous film 3, and the average spacing may have a lower limit of, for example, 1 ⁇ m or greater, 5 ⁇ m or greater, 8 ⁇ m or greater, 10 ⁇ m or greater, 12 ⁇ m or greater or 15 ⁇ m or greater.
  • the upper limit for the average spacing between the convex sections (76, 77, 77a, 77b, 78) may be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 180 ⁇ m or smaller, 170 ⁇ m or smaller, 150 ⁇ m or smaller, 125 ⁇ m or smaller or 100 ⁇ m or smaller, for example.
  • the average spacing between the convex sections may be 1 ⁇ m to 300 ⁇ m, 5 ⁇ m to 250 ⁇ m, 8 ⁇ m to 200 ⁇ m, 10 ⁇ m to 180 ⁇ m, 12 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the separation width W7a between the convex sections can be determined based on the intersections (C, C') of the convex sections, where the convex sections 77a and convex sections 77b cross.
  • the widths (Y6, Y7, Y8) of the convex sections may be uniform or non-uniform.
  • the average lower limit for the widths (Y6, Y7, Y8) of the convex sections may be 1 ⁇ m or greater, 5 ⁇ m or greater, 10 ⁇ m or greater, 15 ⁇ m or greater, 20 ⁇ m or greater or 25 ⁇ m or greater, for example.
  • the average upper limit for the widths (Y6, Y7, Y8) of the convex sections may be 300 ⁇ m or smaller, 250 ⁇ m or smaller, 200 ⁇ m or smaller, 150 ⁇ m or smaller, 100 ⁇ m or smaller, 80 ⁇ m or smaller or 75 ⁇ m or smaller, for example.
  • the average for the widths (Y6, Y7, Y8) of the convex sections may be 1 ⁇ m to 300 ⁇ m, 5 ⁇ m to 250 ⁇ m, 10 ⁇ m to 200 ⁇ m, 15 ⁇ m to 150 ⁇ m, 20 ⁇ m to 100 ⁇ m, 25 ⁇ m to 80 ⁇ m or 30 ⁇ m to 75 ⁇ m, for example.
  • the surfaces of the convex sections (76, 77, 77a, 77b, 78) may also be subjected to plasma treatment by a publicly known method, in order to facilitate adhesion of the cells.
  • fibers that have been fragmented to arbitrary lengths may be used and anchored onto the porous film 3 to form the convex sections (76, 77, 77a, 77b, 78), or the convex sections (76, 77, 77a, 77b, 78) may be provided by contact bonding, or the convex sections (76, 77, 77a, 77b, 78) may be simply placed on the porous film 3.
  • the method of anchoring the convex sections (76, 77, 77a, 77b, 78) onto the porous film 3 may be anchoring onto the porous film 3 in a covalently bonded manner by a publicly known method, or anchoring using an adhesive.
  • the adhesive is preferably a substance that does not affect cell adhesion and has no toxicity for cells, and that is not degraded by the cells. Examples include poly-D-lysine, agarose, glucomannan and ultraviolet curing resins, but any other ones may be used so long as they are substances that produce a similar effect and are able to anchor the fibers onto the porous film.
  • the convex sections (76, 77, 77a, 77b, 78) are preferably anchored onto the porous film 3 so that they do not detach in the cell culture. If a culture vessel 1 is used having the convex sections (76, 77, 77a, 77b, 78) and porous film 3 anchored, then it will be possible to obtain a three-dimensional culture skin sheet that not only is thickened but also has a high barrier function.
  • the material of the cell culturing vessel 1 used for the embodiments of the invention is not particularly restricted so long as it is a material commonly used for cell culturing vessels. Examples include glass, polystyrene, polypropylene and polycarbonate.
  • the form of the cell culturing vessel used for the invention is also not particularly restricted, and it may be in the form of a dish, multi-well plate, flask or the like. Since the cell culturing vessel of the invention has a porous film on a portion of the cell culturing vessel, it is preferably in the form of a cell culture insert.
  • a cell culture insert to be used for the invention is a cell culturing vessel comprising a film having porous holes that are impermeable to cells but permeable to culture solution. Culture solution may also be supplied from the opposite side of the culture surface of the porous film, i.e. the back side of the adhering surface of the adherent cells.
  • the cell culture insert that may be used for the invention may be a commercially available one, or it may be a cell culture insert having a film provided with convex sections directly molded onto the culture surface of a porous film. When a commercially available one is used, the convex sections may be formed on the porous film surface by bonding beads or fibers, for example, or convex sections may be simply placed on the porous film 3 without bonding.
  • mesh-like convex sections of approximately the same diameter as the inside of the cell culture insert 2 (as in Fig. 7(a) , for example) may be fitted into the cell culture insert 2 without bonding, for example.
  • the convex sections may be provided together with or separately from the cell culture insert 2 as a portion of the culture vessel for preparation of the three-dimensional culture skin sheet, and they may be part of a kit to be assembled during culturing.
  • the mean pore size of the porous film is not particularly restricted, and it may be 0.1 ⁇ m to 5.0 ⁇ m, preferably 0.2 ⁇ m to 4.0 ⁇ m and more preferably 0.3 to 3.5 ⁇ m, for example.
  • the material used for the porous film may also be a polyester or polyethylene terephthalate (PET), or polycarbonate, polystyrene or the like, similar to porous films used in the prior art.
  • the culture surface may be coated to facilitate adhesion of the cells onto the cell culturing vessel that has convex sections on the porous film 3.
  • examples include collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (the D-form or L-form) and thrombospondin.
  • the number of seedings of epidermal cells may be according to a publicly known method.
  • seeding of the epidermal cells may be in an amount of 0.01 ⁇ 10 6 to 10.0 ⁇ 10 6 /cm 2 , preferably 0.05 ⁇ 10 6 to 5.0 ⁇ 10 6 /cm 2 and more preferably 0.1 ⁇ 10 6 to 1.0 ⁇ 10 6 /cm 2 .
  • the culture medium (also referred to as "culture solution") to be used for culturing of the epidermal cells may be commonly used culture medium such as KG culture medium, EpilifeKG2 (Kurabo Industries, Ltd.), Humedia-KG2 (Kurabo Industries, Ltd.), assay culture medium (Toyobo), CnT-Prime or Epithelial culture medium (CELLnTEC), and culturing may be carried out at about 37°C for a period of 0 to 14 days.
  • the culture medium used may alternatively be DMEM culture medium (Gibco), or a culture medium comprising a 1:1 mixture of 2-0-a-D-glucopyranosyl-L-ascorbic acid-containing KGM and DMEM.
  • CnT-Prime, 3D barrier medium (CELLnTEC) may be used for three-dimensionalization (overlaying) of the epidermal cells.
  • Other culture media may also be used.
  • the epidermal cells in order to culture the epidermal cells and achieve three-dimensionalization (overlaying) to promote keratinization, it is sufficient to seed the cells in a cell culturing vessel 1 containing a cell culture insert 2, and carry out growth culture. Specifically, the epidermal cells are suspended in medium and seeded onto the cell culture insert 2. In order to contact the culture medium with the outer side of the porous film 3 of the cell culture insert, culture medium is also added to the bottom well 4, and the cell culture insert 2 is immersed and cultured. As a result, the culture medium is supplied to both the top and bottom parts of the epidermal cells, and they are cultured.
  • the epidermal cells on the cell culture insert 2 are preferably cultured for about several days (about 1 to 6 days, and preferably about 2 to 4 days), until confluent or subconfluent.
  • the culture medium inside and outside the cell culture insert is preferably exchanged with CnT-Prime, 3D barrier medium (CELLnTEC), for example. This will further promote three-dimensionalization of the epidermal cells.
  • CELLnTEC 3D barrier medium
  • culturing preferably after 1 to 36 hours, preferably after 6 hours to 24 hours and more preferably after 12 hours to 18 hours, the culture medium inside the cell culture insert alone is removed and the top surfaces of the epidermal cells are exposed to a gas phase for culturing. This promotes three-dimensionalization and keratinization of the epidermal cells, yielding a thicker three-dimensional culture skin sheet.
  • the culturing temperature for each culturing step of the invention may be near the body temperature of the animal source, and specifically for human cells, it is preferably about 33°C to 38°C.
  • the three-dimensional culture skin sheet obtained by the invention in the manner described above can be used as an animal experiment substitute, such as a skin model.
  • a skin model For example, it may be used in a method of evaluating reactivity of skin to chemical substances (for example, cosmetics, industrial products, household goods, drugs, external preparations for skin, and the like).
  • the three-dimensional culture skin sheet of the invention can provide thicker tissue compared to conventional three-dimensional culture skin sheets, it may be used as an effective skin model for basic dermatologic research as well.
  • the three-dimensional culture skin sheet obtained by the invention is thicker than conventional three-dimensional culture skin sheets, it has a higher barrier function from the outside and is also useful as a three-dimensional culture skin sheet for healing of burns and wounds.
  • the cell culturing vessel used for the invention may be a cell culturing vessel in which the convex sections and porous film are separable after completion of culturing.
  • the three-dimensional culture skin sheet produced by the cell culturing vessel is separated from the porous film while the convex sections are still in contact with the base section.
  • the base section of the three-dimensional culture skin sheet can thus be moved while maintaining the concavoconvex structure.
  • the convex sections are preferably a biological substance including collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (the D-form and L-form) or thrombospondin, for example, with insoluble collagen being more preferred.
  • the epidermal cells used for the invention were neonatal derived keratinocytes (hereunder, "keratinocytes”; product name Frozen NHEK (NB), Catalog No.: KK-4009, Kurabo Industries, Ltd.). Subculturing was carried out according to the instructions provided by the vendor.
  • Fig. 9 The results are shown in Fig. 9 .
  • (a) wherein the culture surface of the cell culture insert was untreated, it was only possible to obtain a three-dimensional cultured epidermis with a thickness of about 10 ⁇ m, but with the cell culture insert provided with convex sections (b), thickening and stratum corneum formation of the three-dimensional culture cells were clearly observed.
  • the thickness of the stratum corneum was nearly twice that of the control (a).
  • Fig. 9(b) has partial hollowed sections, this was caused by handling during sample preparation.
  • Fig. 10 shows magnified views of polyester fibers used in the Examples for formation of concavoconvexities in a three-dimensional culture skin sheet of the invention.
  • Table 1 The characteristics of the fibers used for this example are shown in Table 1 below. Table 1 also shows the results that the convex sections formed using them had on formation of the three-dimensional culture skin sheet. The open area ratio was calculated based on the ratio of the area of the regions without mesh-like fibers, to the culture area.
  • Fig. 11A and Fig. 11B show HE staining images of prepared three-dimensional culture skin sheet slices. Although sections with separation of the three-dimensional culture skin sheets and the porous films of the cell culture inserts, curling of the fibers and hollowing can be seen in Fig. 11A and Fig. 11B , these were all artificially created by handling during sample preparation.
  • the barrier function was evaluated according to the following procedure.
  • the method was based on the same principle as the method described in an article by Kumamoto (see Kumamoto J., Tsutsumi M., Goto M., Nagayama M., Denda M., Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo. Arch. Dermatol. Res. 308:49-54, 2016 ).
  • the obtained results were statistically analyzed (ANOVA analysis of variance, followed by Scheffe multiple test analysis), and multiple test results with p ⁇ 0.05, compared to the three-dimensional culture skin sheet obtained by culturing with the film alone, were judged to have significant difference.
  • the three-dimensional culture skin sheet obtained with the cell culture inserts having the No. 255 fabric and No. 300 fabric bonded using an ultraviolet curing resin had significantly lower transepidermal water loss compared to the three-dimensional culture skin sheet without concavoconvexities, demonstrating that a three-dimensional culture skin sheet with a high barrier function had been obtained ( Fig. 12 ).

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EP17815530.5A 2016-06-24 2017-06-23 Dreidimensional gezüchteter hautlappen, zur herstellung davon verwendeter zellzüchtungsbehälter und verfahren zur herstellung eines dreidimensional gezüchteten hautlappens Withdrawn EP3476933A4 (de)

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PCT/JP2017/023302 WO2017222065A1 (ja) 2016-06-24 2017-06-23 三次元培養皮膚シート、その製造に使用するための細胞培養容器及びその製造方法

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WO2019219605A1 (de) * 2018-05-16 2019-11-21 Cellbricks Gmbh Verfahren zur herstellung eines zellkultureinsatzes mit mindestens einer membran

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JP7373833B2 (ja) * 2018-07-09 2023-11-06 学校法人東京女子医科大学 三次元生体組織の培養方法、並びに三次元生体組織培養デバイス及びシステム
JP7506661B2 (ja) 2019-04-04 2024-06-26 株式会社 資生堂 三次元培養皮膚シート、その製造に使用するための細胞培養容器及びその製造方法
JP7304022B2 (ja) * 2019-10-16 2023-07-06 国立研究開発法人理化学研究所 細胞シート製造装置、および細胞シート
JP7493945B2 (ja) 2020-01-28 2024-06-03 ポーラ化成工業株式会社 培養組織の観察方法、培養方法、評価方法及び培養器具
KR102460180B1 (ko) * 2020-11-27 2022-10-27 숭실대학교산학협력단 연령별 구조 및 물성을 갖는 인공피부, 및 이의 제조방법
CN113293097B (zh) * 2021-05-19 2023-03-21 法国介观生物技术有限公司 一种人工基底膜及包含其的细胞培养装置
CN113621554B (zh) * 2021-08-13 2023-05-09 杭州捷诺飞生物科技股份有限公司 采用同一无血清培养基的表皮组织简易制备工艺及其保存

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WO2019219605A1 (de) * 2018-05-16 2019-11-21 Cellbricks Gmbh Verfahren zur herstellung eines zellkultureinsatzes mit mindestens einer membran

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JP6993329B2 (ja) 2022-02-04
JPWO2017222065A1 (ja) 2019-04-11
KR20190020654A (ko) 2019-03-04
EP3476933A4 (de) 2020-03-04
WO2017222065A1 (ja) 2017-12-28
US20190143002A1 (en) 2019-05-16
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